Simon Scheuring, Weill Cornell Medicine Scientific Area : 6 MCB : Molecular and Cellular Biology / 5 IE : Instrumentation and Engineering Project Summary/Abstract (30 lines of text): In the recent years, we have seen tremendous progress in structural biology owing to breakthroughs in 3D- crystallization methodology for X-ray diffraction and improved particle classification algorithms and the development of direct electron detection for cryo-EM. As a result, membrane protein structure resolution is now rather routine and progresses at a pace of almost 2 structures per week. To complement structures, technologies like FRET, EPR and HDX give invaluable insights into the range of dynamics and kinetics of conformational states. All experimental structural and dynamical techniques have however a blind spot: they are poorly adapted to analyze proteins in response to physical stimuli such as force, temperature and voltage. This is particularly regrettable for the case of sensory ion channels that process these physical stimuli, because they are involved in some of the most crucial physiological functions and are implicated in various pathologies. Another technique that is powerful to assess conformational dynamics is high-speed atomic force microscopy (HS-AFM), this approach has two significant advantages: (i) it is also a structural technique, meaning that it provides real-space real-time movies of molecules, and (ii) it operates under physiological and changeable conditions. Thus, the first advantage allows to characterize the structure and conformational changes of the channels at ~1nm lateral, ~0.1nm vertical and ~100ms temporal resolution. While the second advantage opens the experimental tool to the application of external stimuli, (bio)chemical and also, importantly, physical stimuli. In this project, we will develop novel extensions to HS- AFM to take movies of the conformational response of sensory channels to such physical cues. We will expose mechano-sensitive Piezo channels to force, temperature-sensitive TRPV channels to temperature- sweeps, and voltage-gated K+ channels to the direct application of transmembrane voltage, and image the structural changes of these proteins in response to such stimuli. This project will, on the one hand push the limits of HS-AFM technologically and create novel operational modalities of it and such further establish this rather new technology for a wide range of structure-function application in biomedical research, and on the other hand be transformative for the structural biology of sensory ion channels by providing insights into long-standing questions how these biological machines transform such physical stimuli into coordinated conformational dynamics that ultimately lead to channel gating.